BACTERIAL TOXIN-NEUTRALIZING NANOBODIES AND METHODS OF MAKING AND USING THE SAME

Abstract

The presently disclosed subject matter is generally directed to novel nanobodies that can be used to neutralize bacterial toxins in a subject affected with harmful bacteria, such as bacteria associated with anthrax exposure and toxic shock syndrome. The disclosed nanobodies may be capable of binding to antigenic sites that are functionally invisible to larger antibody proteins. Further, the low molecular weight and compact size of the disclosed nanobodies confer thermostability characteristics, such that they are stable in extreme pH conditions and when exposed to proteolytic digestion. Most importantly, the disclosed nanobodies can readily move from the circulatory system into tissues to disrupt the disease process.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/596,144, filed Dec. 8, 2017, the entire content of which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] The presently disclosed subject matter relates generally to immunotherapeutics. More specifically, the presently disclosed subject matter relates to nanobodies that can neutralize bacterial toxins, and methods for using nanobodies to treat subjects exposed to such toxins.

BACKGROUND

[0003] Some disease-causing bacteria produce and release toxins, and these toxins can have a significant role in morbidity and mortality. Although antibiotics can eradicate the infection associated with the bacteria, they cannot neutralize the bacterial toxins released prior to eradication of the infection. Hence, the bacterial toxins can cause damage to host tissues despite successful antibiotic therapy. The need for anti-toxin therapies was particularly exemplified by the 2001 anthrax attacks in the United States. In those attacks, there were 11 documented cases of inhalational anthrax. Despite successful eradication of the infection by the antibiotics in the 5 fatal cases, the disease continued to progress (likely due to the presence of anthrax toxins) and eventually led to the death of the individual (Jernigan J. A., Stephens D. S., Ashford D. A., et al. "Bioterrorism-related Inhalational Anthrax: The First 10 Cases Reported in the United States." Emerg Infect Dis 7 (2001):933-44). Toxic shock syndrome represents another example of a condition where bacterial toxins have been shown to adversely affect an individual. The pyrogenic exotoxins of Group A Streptococci and Staphylococcus aureus can cause an aberrant proliferation of specific T-cell subsets, which leads to significantly increased production of cytokines that cause the shock-like symptoms (Brosnahan A. J. and Schlievert P. M. "Gram Positive Bacterial Superantigen Outside-In Signaling Causes Toxic Shock Syndrome." FEBS J. 278 (2011): 4649-67). Toxic shock syndrome has a high mortality (estimated .about.20,000 cases annually with a 10% mortality rate (Weiss K. A. and Layerdiere M. "Group A Streptococcus Invasive Infections: a Review". Can. J. Surg. 40 (1997): 18-25). It would therefore be beneficial to provide a treatment that neutralizes bacterial toxins in a subject affected with harmful bacteria.

SUMMARY

[0004] [I will update this section once the claims are finalized.]

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The previous summary and the following detailed descriptions are to be read in view of the drawings, which illustrate some (but not all) embodiments of the presently disclosed subject matter.

[0006] FIG. 1a is a representation of a human immunoglobulin G antibody.

[0007] FIG. 1b is a representation of a camelid single domain antibody that comprises variable heavy chain domains and constant heavy chain domains only.

[0008] FIG. 1c is a representation of a shark single domain antibody that comprises variable heavy chain domains and constant heavy chain domains only.

[0009] FIG. 1d is a representation of a nanobody that comprises only the variable heavy chain (V.sub.H) from the antibodies shown in FIG. 1a, 1b, or 1 c.

[0010] FIG. 2a is a schematic illustrating the mechanism of action for internalization of edema factor (EF) and lethal factor (LF) resulting in toxic effects on a cell.

[0011] FIG. 2b is a schematic illustrating the blockade of toxic effects in cells by anti-PA antibodies preventing the internalization of LF and EF.

[0012] FIG. 2c is a schematic illustrating anti-LF and anti-EF nanobody neutralization of extracellular LF and EF.

[0013] FIG. 2d is a schematic illustrating nanobody neutralization of intracellular EF and LF toxins.

[0014] FIG. 3a is a schematic illustrating the mechanism of action of Staphylococcus aureus-triggered toxic shock syndrome.

[0015] FIG. 3b is a schematic illustrating the mechanism of action for toxin neutralization by a nanobody which prevents progression to toxic shock syndrome.

[0016] FIG. 4 is a schematic illustrating a method of phage display selection, phages in E. coli, and analysis in accordance with some embodiments of the presently disclosed subject matter.

[0017] FIG. 5 is a Western blot of biotinylated proteins fixed on streptavidin magnetic beads.

[0018] FIG. 6 is a schematic illustrating antibody phage display selection in accordance with some embodiments of the presently disclosed subject matter.

[0019] FIG. 7 is a bar graph illustrating LF binding during 3 rounds of phage display selection by the output/input ratio.

[0020] FIG. 8 is a bar graph illustrating LF binding results via ELISA assay for 3 rounds of phage display selection.

[0021] FIG. 9 is a schematic illustrating one embodiment of a non-adsorbed phase ELISA assay.

[0022] FIGS. 10a-10f are bar graphs illustrating non-adsorbed ELISA assay results for clones 1-25.

[0023] FIGS. 11a-11f are bar graphs illustrating non-adsorbed V.sub.HH ELSIA assay results for clones 1-25.

[0024] FIG. 12 is an alignment of 10 selected clones to a reference nanobody with CDR region replaced by "x."

[0025] FIG. 13 is a representation of a map of V.sub.FH ref pHEN2.

[0026] FIG. 14 is a Western blot of biotinylated proteins fixed on streptavidin magnetic beads.

[0027] FIG. 15 is a bar graph illustrating EF binding during 3 rounds of phage display selection by the output/input ratio.

[0028] FIG. 16 is a bar graph illustrating EF binding results via ELISA assay for 3 rounds of phage display selection.

[0029] FIGS. 17a-17g are bar graphs illustrating non-adsorbed ELISA assay results for clones 26-66.

[0030] FIGS. 18a-18g are bar graphs illustrating non-adsorbed ELISA IPTG assay results for clones 26-66.

[0031] FIG. 19 is an alignment of 10 clones selected from clones 26-66 compared to a reference nanobody with CFR region replaced by "x."

[0032] FIG. 20 is a schematic illustrating phage display selection, GAP repair, and yeast clone production in accordance with some embodiments of the presently disclosed subject matter.

[0033] FIG. 21 is a Western blot of biotinylated proteins fixed on streptavidin magnetic beads.

[0034] FIG. 22 is a bar graph illustrating TSST1 binding during 3 rounds of phage display selection by the output/input ratio.

[0035] FIG. 23 is a bar graph illustrating TSST1 binding results via ELISA assay for 3 rounds of phage display selection.

[0036] FIG. 24 is a bar graph illustrating V.sub.HHs control for ELISA assay.

[0037] FIG. 25 is an alignment of 10 clones selected from clones 67-94 compared to a reference nanobody with CFR region replaced by "x."

DETAILED DESCRIPTION

[0038] The presently disclosed subject matter is introduced with sufficient details to provide an understanding of one or more particular embodiments of broader inventive subject matters. The descriptions expound upon and exemplify features of those embodiments without limiting the inventive subject matters to the explicitly described embodiments and features. Considerations in view of these descriptions will likely give rise to additional and similar embodiments and features without departing from the scope of the presently disclosed subject matter.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently disclosed subject matter pertains. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described.

[0040] Following long-standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in the subject specification, including the claims. Thus, for example, reference to "a nanobody" can include a plurality of such nanobodies, and so forth.

[0041] Unless otherwise indicated, all numbers expressing quantities of components, conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the instant specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the scope of the presently disclosed subject matter.

[0043] The presently disclosed subject matter is generally directed to novel nanobodies that can be used to neutralize bacterial toxins in a subject infected with harmful bacteria. As used herein, the term "nanobody" refers to a single-domain antibody fragment that comprises the unique structural and functional properties of naturally-occurring heavy chain only antibodies. Thus, nanobodies are antibody-derived therapeutically active proteins. Like whole antibodies, nanobodies are able to bind selectively to a specific antigen. FIG. 1a illustrates human immunoglobulin G (IgG) antibody that includes a light chain with two domains, the variable light chain domain (V.sub.L) and the constant light chain domain (C.sub.L). IgG antibody further comprises a heavy chain with four domains, one variable heavy chain domain (V.sub.H) and three constant heavy chain domains (C.sub.H1, C.sub.H2, and C.sub.H3).

[0044] In some embodiments, the disclosed nanobodies can be camelid-derived (i.e., obtained from members of the camelid family) that specifically bind to an antigen or a fragment thereof. Suitable members of the camelid family can include (but are not limited to) Camelus bactrianus and Camelus dromaderius, as well as llama species (Vicugna pacos, Lama glama and Vicugna vicugna). Advantageously, various IgG antibodies from the camelid family lack light chains as illustrated in FIG. 1b, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains. The small single variable region (identified as V.sub.H) of the camelid domain can be obtained by genetic engineering to yield a nanobody having high affinity for a desired target. Such methods are well known in the art. Nanobodies engineered from heavy-chain antibodies found in camelids are termed "V.sub.HH fragments."

[0045] Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies (called VNAR) can be obtained. Suitable cartilaginous fishes can include (but are not limited to) sharks. Particularly, certain species of shark produce functional antigen-binding heavy chain antibodies naturally devoid of the light chain. As shown in FIG. 1c, the shark antibody comprises five constant domains (C.sub.H1, C.sub.H2, C.sub.H3, C.sub.H4, C.sub.H5) and one variable domain (V.sub.H). As with camelid-derived nanobodies, the variable domain (V.sub.H) of the cartilaginous fish antibody can be isolated using genetic engineering to yield a nanobody having high affinity for a target.

[0046] As an alternative, the dimeric variable domains from common human or murine IgG can be split into monomers (V.sub.H and V.sub.L fragments). Such methods are well known to those of ordinary skill in the art.

[0047] As shown in FIG. 1d, the illustrated nanobody comprises only the variable heavy chain (V.sub.H) of the antibody of FIG. 1a, FIG. 1b, or FIG. 1c.

[0048] In some embodiments, the disclosed nanobodies (e.g., camelid-derived or shark-derived) can be humanized. As used herein, the term "humanized nanobody" refers to a nanobody wherein one or more amino acid residues of the naturally occurring amino acid sequence of the antibody domain has been replaced by one or more amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional chain human antibody. Methods of humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions can be selected such that the resulting humanized nanobodies retain favorable properties, such as binding specificity. Those skilled in the art would be able to determine and select suitable humanizing substitutions and/or suitable combinations of humanizing substitutions.

[0049] Advantageously, nanobodies have a molecular weight that is approximately 1/10 that of a human IgG molecule. As a result, nanobodies exhibit desirable characteristics while maintaining the target specificity of antibodies (Harmsen M. M. and De Haard H. J., "Properties, Production, and Applications of Camelid Single-Domain Antibody Fragments". Appl Microbiol Biotechnol 77 (2007): 13-22). For example, one benefit of the small size of the disclosed nanobodies is the ability to bind to antigenic sites that are functionally invisible to larger antibody proteins. Further, the low molecular weight and compact size of the disclosed nanobodies confer thermostability characteristics, such that they are stable in extreme pH conditions (e.g., pH 1-3 and/or 11-14) and when exposed to proteolytic digestion. Most importantly, the disclosed nanobodies can readily move from the circulatory system into tissues. Table 1 summarizes several key differences between nanobodies and antibodies.

TABLE-US-00001 TABLE 1 Comparison of Antibody and Nanobody Characteristics.sup.a Antibody Nanobody Molecular Weight 150-160 kDa 12-15 kDa Specificity Similar Similar Tissue Distribution Limited Better Intracellular Distribution Poor Good Distribution Across Blood- Negligible Some Brain Barrier Ability to Target Enzyme Limited Better Active Sites Elimination Half-Life About 20 days.sup.b About 2 hours Subject to Renal No Yes Elimination Heat Resistance Limited Better Stability to Urea/Detergent Limited Better Resistance to Gastric Limited Better.sup.c Acid/Proteases Complement Fixation Yes No Sources Human, murine, most Camelids, mammals Cartilaginous fishes Ability to be Fully Yes Yes Humanized Production Systems Usually mammalian due Yeast or bacteria to post-expression can be used modifications e.g., glycosylation) Immunogenicity Potential Variable Low.sup.d .sup.aBased on: Harmsen M M and De Haard H J., 2007. .sup.bFor a typical human antibody; half-lives may be shorter for chimeric antibodies or antibodies of nonhuman origin. .sup.cStrategies have been reported for improving resistance (Harmsen M M and De Haard H J., 2007) .sup.dBased on small size, stable behavior, rapid clearance from blood, and a sequence sharing a high degree of identity with human V.sub.H; strategies exist for humanization.

[0050] In some embodiments, the presently disclosed subject matter includes isolated nanobodies that specifically bind to B. anthracis toxins (such as lethal factor (LF) and edema factor (EF)) and S. aureus toxins (such as toxic shock syndrome toxin 1 (TSST-1)). However, it should be appreciated that the presently disclosed subject matter is not limited and can include nanobodies that neutralize a wide variety of bacterial toxins.

[0051] The most severe form of anthrax infection occurs following inhalation of B. anthracis spores that germinate within macrophages as they travel to the draining mediastinal lymph nodes in a subject. Although the bacterial replication (bacteremia) can be controlled by administration of appropriate antibiotics, the bacterial toxins exert deleterious effects on the cells within the body. As a result, substantial pathology and high mortality occurs in infected individuals. Because they have no direct effect on the toxins, antibiotics are unable to treat the effects of the toxins.

[0052] B. anthracis produces 3 toxins, which include a binding moiety, protective antigen (PA), and 2 enzymatic moieties, edema factor (EF) and lethal factor (LF) (Ascenzi P., Visca P., Ippolito G., et al. "Anthrax toxin: A Tripartite Lethal Combination." FEBS Letters 531 (2002): 384-8; Inglesby T. V., O'Toole T., Henderson D. A., et al. "Anthrax as a Biological Weapon, 2002: Updated Recommendations for Management." JAMA 287 (2002): 2236-52). The mechanism of action of B. anthracis is illustrated in the schematic of FIG. 2a. Particularly, the bacterial PA antigen first binds to its cell surface receptor and is cleaved by a membrane-bound furin-like protease leaving a 63 kDa fragment bound to the cell. The bound antigen fragment multimerizes into a heptameric barrel structure and exposes a site to which edema factor (EF) and lethal factor (LF) bind with high affinity. Internalization of the complex by receptor-mediated endocytosis is followed by the formation of a membrane-spanning pore. The bound EF and LF proteins are then translocated through the pore to the cytosol of the cell to exert a toxic effect.

[0053] FIG. 2b is a schematic illustrating immunity to anthrax infection conferred by stimulating the body to produce antitoxin antibody (Friedlander A. M., Welkos S. L., Ivins B. E. "Anthrax Vaccines". Curr. Top. Microbiol. Immunol. 271 (2002): 33-60) or by passive immunization, e.g. administration of an anti-PA antibody. As shown, the antitoxin antibodies prevent PA binding to cell surface receptors, thereby preventing internalization of LF and EF. However, the toxic effects in cells that have internalized LF and EF are still present, leading to cell death. To this end, multiple published studies have shown that antibiotics or anti-PA antibodies can be 100% effective in the treatment of inhalational anthrax if administered early enough after exposure to B. anthracis spores. However, when treatment is delayed until after the onset of symptoms, not all treated subjects survive (Hou A. W., Morrill A. M. "Obiltoxaximab: Adding to the Treatment Arsenal for Bacillus anthracis Infection." Ann. Pharmacother. 51 (2017): 908-13; Huang, E., Pillai, S. K., Bower W. A., et al. "Antitoxin Treatment of Inhalation Anthrax: A Systematic Review." Health Secur. 13 (2015): 365-77; Rubinson L., Corey A., Hanfling D. "Estimation of Time Period for Effective Human Inhalational Anthrax Treatment Including Antitoxin Therapy". PLOS. Curr. Outbreaks. 1: (2017)).

[0054] Two monoclonal anti-PA antibodies (Raxibacumab and Obiltoxaximab) that afford passive immunization have been approved by the Food and Drug Administration (Tsai C-W, and Morris S. "Approval of Raxibacumab for the Treatment of Inhalation Anthrax Under the U.S. Food and Drug Administration `Animal Rule.`" Front. Microbiol. 6 (2015): 1320; Greig S. L. "Obiltoxaximab: First Global Approval." Drugs. 76 (2016): 823-30). Anti-PA bispecific V.sub.HH-based agent (2 linked anti-PA nanobodies) have also been reported as providing prophylactic protection from anthrax (Moayeri M., Tremblay J. M., Debatis M., et al. "Adenoviral Expression of a Bispecific V.sub.HH-Based Neutralizing Agent that Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice". Clin. Vaccine Immunol. 23 (2016): 213-8). The latter provides support for the assertion that using nanobodies to neutralize LF/EF should have a therapeutic benefit.

[0055] The mortality observed with delayed post-exposure treatment is believed to result from the deleterious effects of intracellular LF and EF, which antibiotics and anti-PA antibodies do not neutralize. Thus, anti-LF and anti-EF nanobodies have the potential to protect against extracellular toxins, as shown in the schematic of FIG. 2c. Particularly, anti-LF and anti-EF nanobodies neutralize extracellular EF and LF, thereby preventing internalization of LF and EF. As a result, the cells are spared any toxic effects. As illustrated in the schematic of FIG. 2d, in cells that have already internalized bacterial toxins, nanobodies enter the cytosol to neutralize intracellular toxins, thereby ceasing the deleterious effects that lead to cell death.

[0056] Thus, nanobodies offer an advantage over existing therapies and hold the potential to prevent the mortality associated with delayed post exposure treatment of bacterial infections, such as inhalational anthrax. However, use of the disclosed nanobodies to treat, prevent, and/or ameliorate bacterial infections is not limited to B. anthracis infections. For example, the disclosed nanobodies are also believed to be beneficial in the treatment of toxic shock syndrome. Toxic shock syndrome is primarily caused by Streptococcus pyogenes or S. aureus (Low, D. E. "Toxic Shock Syndrome: Major Advances in Pathogenesis, but not Treatment." Critical Care Clinics. 29 (2013): 651-75). Antibiotics are the first-line treatment for toxic shock syndrome, but the mortality rate remains unacceptably high (Lappin E., Ferguson A. J. "Gram-Positive Toxic Shock Syndromes." Lancet Infect. Dis. 9 (2009): 281-90). Bacterial toxin-mediated activation of the inflammatory cascade is believed to be the primal event in toxic shock syndrome pathogenesis.

[0057] The toxic shock syndrome toxin 1 (TSST-1) of S. aureus is considered to have a pivotal role in the pathogenesis of toxic shock syndrome (Schlievert P. M., Osterholm M. T., Kelly J. A., et al. "Toxin and Enzyme Characterization of S. aureus Isolated from Patients with and without Toxic Shock Syndrome." Ann. Intern. Med. 96 (1982): 937-40). The schematic of FIG. 3a illustrates the role of TSST-1 as instigator of the cascade that manifests as toxic shock syndrome. Specifically, S. aureus releases TSST-1, which binds to major histocompatibility complex (MHC) Class II receptors on antigen presenting cells (APC) and receptors on T cells, resulting in proliferation of T cells and subsequent release of cytokines. The symptoms of toxic shock syndrome develop as a result of the proliferation and cytokine release. FIG. 3b illustrates a schematic wherein an anti-TSST-1 nanobody blocks the instigator action by preventing TSST-1 from binding to cell surface receptors, preventing the proliferation/cytokine release cascade.

[0058] TSST-1 is just one of many superantigens (Proft T., Fraser J. D. "Bacterial Superantigens." Clin. Exp. Immunol. 133 (2003): 299-306; Dinges M. M., Orwin P. M., Schlievert P. M. "Exotoxins of Staphylococcus aureus." Clin. Microbiol. Rev. 13 (2000): 16-34). TSST-1 from S. aureus shares similar biological activity with the staphylococcal enterotoxins (SE) and streptococcal pyrogenic exotoxins (SPE), although the amino acid sequence of TSST-1 differs from these two classes of toxins (Proft and Fraser, 2003). More than 20 SE have been identified (Pinchuk I. V., Beswick E. J., Reyes V. E. "Staphylococcal Enterotoxins." Toxins (Basel) 2 (2010): 2177-97), and at least 11 SPE are known (Proft and Fraser, 2003). Given the relatively unique structure of TSST-1 and its primary role in the development of toxic shock syndrome, it is believed that anti-TSST-1 nanobodies can be used to help prevent development of toxic shock syndrome.

[0059] However, it should be appreciated that neutralization of other superantigens can also be desirable based on the known synergistic effects of TSST-1 with the SE/SPE family of toxins (Smith R. J., Schlievert P. M., Himelright I. M., et al. "Dual Infections with Staphylococcus aureus and Streptococcus pyogenes Causing Toxic Shock Syndrome. Possible Synergistic Effects of Toxic Shock Syndrome Toxin 1 and Streptococcal pyrogenic Exotoxin C." Diagn. Microbiol. Infect. Dis. 19 (1994): 245-7; Stevens D. L., Tanner M. H., Winship J., et al. "Severe Group A Streptococcal infections Associated with a Toxic Shock-like Syndrome and Scarlet Fever Toxin A." N. Engl. J. Med. 321 (1989): 1-7). Gram negative bacterial endotoxins and the pyrogenic toxins can work synergistically to produce septic shock (Bannan J., Visvanathan K., Zabriskie J. B. "Structure and Function of Streptococcal and Staphylococcal Superantigens in Septic Shock." Infect. Dis. Clin. North Am. 13 (1999): 387-96).

[0060] In some embodiments, the presently disclosed subject matter relates generally to polypeptides that comprise a V.sub.HH of a camelid heavy chain nanobody or a VNAR domain of a cartilaginous fish heavy chain antibody. In some embodiments, the polypeptides bind to a target selected from B. anthracis edema factor (EF), B. anthracis lethal factor (LF), and S. aureus toxic shock syndrome toxin/(TSST-1) to thereby neutralize the target.

[0061] The disclosed nanobodies are thus capable of selectively binding to EF, LF, and/or TSST-1. In some embodiments, the disclosed nanobodies are specific for EF, LF, and/or TSST-1 (e.g., do not bind to any other molecules). In some embodiments, the presently disclosed subject matter is directed to a nanobody directed against LF comprising the amino acid sequence of one or more of the amino acid sequences of SEQ ID NO: 1-10 and/or the nucleotide sequences set forth in SEQ ID NO: 31-40. In some embodiments, the presently disclosed subject matter is directed to a nanobody directed against EF comprising the amino acid sequence of one or more of the amino acid sequences of SEQ ID NO: 11-20 and/or the nucleotide sequences set forth in SEQ ID Nos: 41-50. In some embodiments, the presently disclosed subject matter is directed to a nanobody directed against TSST-1 comprising the amino acid sequence of one or more of the amino acid sequences of SEQ ID NO: 21-30 and/or the nucleotide sequences set forth in SEQ ID NO: 51-60.

[0062] Thus, in some embodiments, the disclosed nanobody can comprise the amino acid sequence of one or more of SEQ ID NOs: 1-30. In some embodiments, the disclosed nanobody can have an amino acid sequence with a sequence identity of at least about 60% with the amino acid sequence of one or more of SEQ ID Nos: 1-30. Thus, the presently disclosed subject matter can include a nanobody comprising an amino acid sequence with a sequence identity of at least about 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% with one or more of SEQ ID NOs: 1-30. Alternatively or in addition, the nanobody can comprise a functional fragment of the amino acid sequence of one or more of SEQ ID NOs:1-30.

[0063] In some embodiments, the disclosed nanobody can comprise the nucleotide sequence of one or more of SEQ ID NOs: 31-60. In some embodiments, the disclosed nanobody can have a nucleotide sequence with a sequence identity of at least about 60% with the nucleotide sequence of one or more of SEQ ID Nos: 31-60. Thus, the presently disclosed subject matter can include a nanobody comprising a nucleotide sequence with a sequence identity of at least about 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% with one or more of SEQ ID NOs: 31-60. Alternatively or in addition, the nanobody can comprise a functional fragment of the nucleotide sequence of one or more of SEQ ID NOs:31-60.

[0064] The term "sequence identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percentage of sequence identity" refers to the valued determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include (but are not limited to) 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%.

[0065] Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and can be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and/or by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG.RTM. Wisconsin Package.RTM. (Accelrys Inc., Burlington, Mass.).

[0066] The disclosed nanobodies can be modified, e.g., by the covalent attachment of any type of molecule provided that the covalent attachment does not prevent the nanobody from binding to its antigen. Examples of suitable modifications can include (but are not limited to) glycosylation, acetylation, pegylation, phosphorylation, amidation, and the like. In some embodiments, the nanobodies can be derivatized by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other proteins, and the like. In some embodiments, the nanobodies can comprise post-translational moieties that improve upon nanobody activity or stability, such as (but not limited to) sulfur, methyl, carbohydrate, phosphorus, and the like.

[0067] It should be appreciated that functionally conservative variants of the disclosed nanobodies are also included within the scope of the presently disclosed subject matter. The term "functionally conservative variant" refers to variants in which a given amino acid is substituted without altering the global conformation and the function of the nanobody, including a replacement of an amino acid with another having similar properties (for example polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, presence of an aromatic group, etc). The amino acids having similar properties are well known to one skilled in the art. The functionally conservative variants preserve their capability of binding LF, EF, and/or TSST-1. In some embodiments, functionally conservative variants can have a bond affinity with LF, EF, and/or TSST-1 equal or increased relatively to the corresponding nanobody.

[0068] By knowing the nucleotide and amino acid sequences of a nanobody of interest, one skilled in the art would be capable of producing the nanobodies according to the presently disclosed subject matter by conventional techniques. For example, the nanobodies can be synthesized by using a well-known synthesis method in a solid phase (Merrifield (19962) Proc. Soc. Ex. Boil. 21:412; Merrifield (1963) J. Am. Chem. Soc. 85:2149; Tam et al. (1983) J. Am. Chem. Soc. 105:6442), by using a commercially available peptide synthesis apparatus, (such as the one made by Applied Biosystems, Foster City, Calif.) and by following the instructions of the manufacturer. Alternatively, the nanobodies can be synthesized with recombinant DNA techniques well-known in the art (e.g., Maniatis et al. (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, NY, 51-54 and 412-430). For example, the nanobodies can be obtained as DNA expression products after incorporating DNA sequences coding for the polypeptide of interest in expression vectors and introducing these vectors in suitable prokaryotic or eukaryotic hosts that will express the polypeptide of interest, from which they may then be isolated by using techniques well known to one skilled in the art. A nanobody comprising or consisting of the sequence of amino acids selected from SEQ ID NOs: 1-30 and/or the sequence of nucleotides selected from SEQ ID NO: 31-60 is also included within the scope of the presently disclosed subject matter.

[0069] The disclosed nanobodies can generally be used to treat, prevent, and/or ameliorate a condition characterized by the presence of one or more bacterial toxins. For example, in some embodiments, the disclosed nanobodies can inhibit or prevent binding of bacterial toxins to their receptors. Alternatively or in addition, the disclosed nanobodies can interrupt a bacterial toxin pathway to prevent translocation of the bacterial toxins into a cell. Further, the disclosed nanobodies can modulate the signaling, enzymatic activity, and/or biological mechanisms mediated by one or more bacterial toxins. Thus, the disclosed nanobodies can be used for the prevention and treatment of bacterial toxin-related conditions, such as anthrax exposure and/or toxic shock syndrome.

[0070] In some embodiments, the disclosed nanobodies can bind to a bacterial toxin or fragment thereof, such as (but not limited to) B. anthracis edema factor (EF), B. anthracis lethal factor (LF), and/or S. aureus toxic shock syndrome toxin1 (TSST-1) to neutralize the effects of the toxin. As used herein, the term "neutralize" refers to the ability of a nanobody to inhibit activity of an antigen to which the nanobody binds. Thus, in some embodiments, the disclosed nanobodies can be anti-LF, anti-EF, or anti-TSST1 nanobodies, and are specific for LF, EF, and TSST-1, respectively. As a result, the deleterious conditions caused by the bacterial toxins are ameliorated or eliminated. The nanobodies can neutralize the LF, EF, and/or TSST-1 such that the toxins are unable to cause physiological damage to the cell.

[0071] The presently disclosed subject matter therefore provides for a pharmaceutical composition comprising any of the nanobodies described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be provided as substantially free from any etiologic agents, infectious agents (particularly those infectious for the subject to which the composition is to be administered, e.g., a human), human blood components, and the like. In some embodiments, the nanobodies and associated pharmaceutical compositions are substantially free from any contaminants normally found in a mammalian or bacterial cell extract or preparations.

[0072] When the disclosed nanobodies are humanized, they are significantly less immunogenic and more therapeutically effective and useful when administered to human patients than non-humanized nanobodies.

[0073] In some embodiments, the presently disclosed subject matter comprises a pharmaceutical composition that includes the disclosed nanobodies. The disclosed nanobodies can be administered to a subject as a pharmaceutical preparation. Particularly, the pharmaceutical preparation can comprise at least one isolated nanobody polypeptide and optionally at least one physiologically acceptable carrier. The term "carrier" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the nanobodies, such as (but not limited to) sterile water, aqueous buffers (e.g., phosphate-buffered saline, Ringer's solutions, dextrose solution, Hank's solution), buffered saline, ethanol, polyol, glycerol, alcohol (e.g., ethanol), and/or glycol (e.g., propylene glycol). The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, and/or organism. As would be known to those of ordinary skill in the art, the characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers can include (but are not limited to) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well known in the art.

[0074] The pharmaceutical preparations can be formulated and administered in any suitable manner known or used in the art. See, for example, Remington's Pharmaceutical Sciences, 18.sup.th Ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005), the entire contents of which are hereby incorporated by reference. For example, the pharmaceutical preparations can be formulated for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration), oral administration, and/or for topical administration (e.g. transdermal or intradermal).

[0075] In addition to the carrier, the disclosed formulation can optionally comprise one or more additives in an amount of from about 0.01-5 weight percent. The additives can include one or more binders, thickeners, softeners, dispersions, emulsifiers, preservatives, lubricants, enzymes, sweeteners, perfumes, pigments, pH adjusting agents, fillers, humectants, absorption accelerators, wetting agents, absorbents, and the like. Such additives are well known to those of ordinary skill in the art. In some embodiments, the additives and/or carriers are inert and/or non-reactive with the nanobodies.

[0076] The pharmaceutical composition can further optionally comprise one or more additional therapeutic agents. Any suitable therapeutic agent can be used, such as (but not limited to) an antibiotic (e.g., ciprofloxacin, doxycycline, chloramphenicol, clindamycin, tetracycline, rifampin, and/or vancomycin), a vaccine, an antibody, and/or a second nanobody. In some embodiments, the compositions include a combination of multiple (e.g., two or more) anti-EF, anti-LF, and/or anti-TSST-1 nanobodies, or antigen-binding fragments thereof.

[0077] The compositions can be administered to the subject using any known administration route. For example, in some embodiments, the nanobodies can be administered orally (through the mouth), sublingually (under the tongue), topically (absorbed through the skin), nasally (through the nasal passages), intravenously (injected into a vein), subcutaneously (under the skin), parentally (injection) and/or by inhalation (by breathing through the airway). The nanobodies can therefore be formulated as capsules, cachets, pills, tablets, lozenges, powders, granules, inhalant, or as a solution or a suspension in an aqueous or non-aqueous liquid. Depending on the route of administration, the nanobody can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0078] The nanobody formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nanobody) that can be combined with a carrier material to produce a single dosage form will generally be an amount that produces a therapeutic effect. In some embodiments, the formulation can comprise about 1-99 weight percent active ingredient (e.g., 1-99, 2-90, 5-80, 10-70, 25-60, 30-50 weight percent) and about 99-1 weight percent carrier (e.g., 99-1, 98-10, 95-20, 90-30, 75-40, or 70-50 weight percent).

[0079] Actual dosage levels of the nanobodies in the disclosed pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compositions, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0080] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the nanobody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0081] The term "therapeutically effective" in reference to dose of a nanobody refers to an amount that reduces a sign or a symptom associated with anthrax infection, anthrax toxin toxicity, a sign or a symptom associated with toxic shock syndrome infection, and/or toxic shock syndrome toxicity by at least about 20%, about 40%, about 60%, about 80%, or by complete reduction relative to untreated subjects. In some embodiments, a therapeutically effective amount of a nanobody is an amount sufficient to prevent death in a subject exposed to anthrax and/or toxic shock syndrome. The ability of a nanobody to reduce signs and/or symptoms (including preventing death) associated with anthrax and/or toxic shock syndrome infection can be evaluated in an animal model system predictive of efficacy of the nanobody in treating human anthrax and/or toxic shock syndrome infection.

[0082] Alternatively, a therapeutically effective amount of a nanobody can be evaluated by examining the ability of the nanobody to neutralize an anthrax toxin or toxic shock syndrome toxin in vitro in a toxin neutralization assay. Such assays are well known in the art. One of ordinary skill in the art would be able to determine such therapeutically effective amounts based on factors such as the subject's size, the severity of the signs and/or subject's symptoms, and/or the particular composition or route of administration selected.

[0083] The presently disclosed subject matter also provides for a method of treating or reducing the toxicity of anthrax toxin and/or toxic shock syndrome toxin. Such methods find application both in vivo and in vitro. For in vivo methods, an anti-anthrax (e.g., anti-EF or anti-LF) or anti-toxic shock syndrome (e.g., anti-TSST-1) nanobody is administered to a subject. For in vitro methods, the nanobody is contacted with a material suspected of having anthrax toxin or toxic shock syndrome toxin (e.g., as a material suspected of being contaminated with anthrax toxin) in an amount effective to provide for neutralization of toxin by the nanobody.

[0084] Any subject having or susceptible to anthrax toxin or toxic shock syndrome toxin exposure can be treated according to the methods disclosed herein. In some embodiments, the subject is infected with B. anthracis and/or S. aureus, has been exposed to anthrax or toxic shock syndrome toxin, or is at risk of infection or exposure.

[0085] In related embodiments, the disclosure provides methods for detecting the presence of B. anthracis and/or S. aureus in a sample, such as a biological sample (e.g., blood from an anthrax-infected subject). The method includes contacting a sample suspected of containing B. anthracis toxin and/or S. aureus toxin with one or more of the disclosed nanobodies (e.g., the anti-LF, anti-EF, and/or anti-TSST-1 nanobodies of SEQ ID NOs: 1-30). The contacting occurs under conditions to allow for formation of a specific nanobody-antigen complex. The presence or absence of the complex is then detected, wherein the presence of the complex indicates the presence of a nanobody-antigen complex. The presence of the nanobody-antigen complex indicates the presence of B. anthracis toxin and/or S. aureus toxin in the sample. For example, a blood or tissue sample can be removed from a subject and contacted with a nanobody under conditions which allow detection of anthrax EF and/or LF in the sample to diagnose an anthrax infection in the subject. Similarly, detection of TSST-1 in a sample can be used to diagnose toxic shock syndrome infection in the subject.

[0086] Suitable immunodetection techniques include (but are not limited to) both in vitro methods and in vivo (imaging) methods. Where the methods are in vitro, the biological sample can be any sample in which a selected antigen is present (e.g., blood samples (including whole blood, serum, etc.), tissues, whole cells (e.g., intact cells), tissue or cell extracts, as well as samples obtained from other sources (e.g., packages, letters, food products, and the like). Assays can take a wide variety of forms, such as competition, direct reaction, or sandwich type assays. Exemplary assays include Western blots; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, and the like. The reactions generally include detectable labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between antigen in the sample and the nanobody.

[0087] The term "subject" refers to a mammal, such as a human. However, in some embodiments, the subject can include (but is not limited to) domesticated animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, sheep, horses, and the like), and laboratory animals (e.g., rats, mice, monkeys, and the like).

[0088] Therefore, the disclosed nanobodies offer an advantage over existing therapies and hold the potential to prevent the harmful effects and/or death associated with delayed post-exposure treatment of a variety of bacterial infections.

EXAMPLES

[0089] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Materials and Methods

Phage ELISA IPTG Plaque Assay

[0090] Preculture:

[0091] 96 well Costar.RTM. plates (available from Corning, Inc. Corning, N.Y.) with a volume of 200 .mu.l/well were used. Controls wells on each plate were as follows: A12-empty (control for contamination); A6-control anti 1329v4-A220 (positive control); B6-R3TF3 anti-GFP (negative control). The plates were incubated with agitation at 37.degree. C. overnight in 2.times.TY media (1.6% tryptone, 1% yeast extract, 0.5% NaCl) with 2% glucose, 100 .mu.g/ml Ampicillin. The box was kept in 30% glycerol (stock at -20.degree. C.).

[0092] Culture:

[0093] In a new plate, the preculture was diluted 1/100 (6 .mu.l of preculture for 600 .mu.l of medium) in 2.times.TY media with 0.1% glucose and 100 .mu.g/ml ampicillin. The plate was incubated for 90 minutes at 37.degree. C. with agitation. Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) was added at final concentration of 1 mM (stock: 840 mM). The plate was incubated overnight at 30.degree. C. with agitation. The plate was spun at 3300 rpm for 10 minutes, and the supernatants were retained.

[0094] ELISA:

[0095] Coating was performed using Nunc.RTM. Maxisorp.RTM. plates for 2 hours at room temperature or overnight at 4.degree. C. with 12.5 .mu.l/well of Avidin at 20 .mu.g/ml in PBS. The plates were washed with 50 .mu.l PBS+0.1% Tween, and washed with 50 .mu.l PBS. 12.5 .mu.l of biotinylated antigen (5 .mu.g/ml) in PBS (phosphate buffered saline) was loaded into each well of the plates. The plates were incubated 2 h at room temperature or overnight at 4.degree. C. Saturation: 50 .mu.l/well PBS 0.1% Tween 2% milk were incubated for 1 hour at room temperature or overnight at 4.degree. C. 3 washes with 50 .mu.l of PBS+0.1% Tween were performed and 3 washes with 50 .mu.l PBS were performed.

[0096] The phages were pre-incubated for 20 minutes at room temperature. 48 .mu.l of phages and 12 .mu.l PBS+0.1% Tween+2% milk were added to each well. The phages were incubated 90 minutes at room temperature. The wells were then washed once with 50 .mu.l of PBS+0.1% Tween and washed twice with 50 .mu.l PBS.

[0097] 12.5 .mu.l/well Myc was added at a dilution of 1/500 in PBS+0.1% Tween+2% milk, and was incubated for 1 hour at room temperature. Each well was then washed with 50 .mu.l of PBS+0.1% Tween and washed twice with 50 .mu.l PBS.

[0098] 12.5 .mu.l/well secondary antibody anti-mouse HRP was added to each well at a dilution of 1/5000 and incubated for 1 hour at room temperature. The wells were washed 3.times. with 50 .mu.l of PBS+0.1% Tween and 3 washes with 50 .mu.l PBS.

[0099] Detection: TMB (transparent plates) or ECL (White plates), 25 .mu.l/well TMB (3,3',5,5''-Tetramethylbenzidine) was added. The reaction was stopped by adding 25 .mu.l of sulfuric acid 2N (1 M). A spectrometer reading at DO=450 nm was then taken.

[0100] Phage ELISA Plaque Assay

[0101] Preculture:

[0102] performed as set forth above for the Phage ELISA IPTG plaque.

[0103] Culture:

[0104] In a new plate, the preculture was diluted 1/100 (6 .mu.l of preculture for 600 .mu.l of medium) in 2.times.TY media with 0.1% glucose and 100 .mu.g/ml ampicillin. The plate was incubated for 2.5 hours at 37.degree. C. with agitation. Phage helper was added from a stock at 6.times.10.sup.13/mL (4.times.10.sup.9/well). The plate was incubated 30 minutes at 37.degree. C. without agitation. The plate was spun at 2500 rpm for 5 minutes, and the supernatants were removed by pouring the plate. Pellets were resuspended in 1 mL 2.times.TY medium+ampicillin (100 ug/mL); Kara 50 ug/mL without glucose. The plate was incubated overnight at 30.degree. C. with agitation. The plate was re-spun for 10 minutes at 3300 rpm and the supernatants were retained.

[0105] ELISA:

[0106] Coating was performed using Nunc.RTM. Maxisorp.RTM. plates for 2 hours at room temperature or overnight at 4.degree. C. with 12.5 .mu.l/well of Avidin at 20 .mu.g/ml in PBS. The plates were washed with 50 .mu.l PBS+0.1% Tween, and washed with 50 .mu.l PBS. 12.5 .mu.l of biotinylated antigen (5 .mu.g/ml) in PBS (phosphate buffered saline) was loaded into each well of the plates. The plates were incubated 2 h at room temperature or overnight at 4.degree. C. Saturation: 40 .mu.l/well PBS 0.1% Tween 2% milk were incubated for 1 hour at room temperature or overnight at 4.degree. C. 3 washes with 50 .mu.l of PBS+0.1% Tween were performed and 3 washes with 50 .mu.l PBS were performed.

[0107] The phages were pre-incubated for 20 minutes at room temperature. 48 .mu.l of phages and 17 .mu.l PBS+0.1% Tween+2% milk were added to each well. The phages were incubated 120 minutes at room temperature. The wells were then washed 3.times. with 50 .mu.l of PBS+0.1% Tween and washed 3.times. with 50 .mu.l PBS.

[0108] 12.5 .mu.l/well Myc was added at a dilution of 1/500 in PBS+0.1% Tween+2% milk, and was incubated for 1 hour at room temperature. Each well was then washed with 50 .mu.l of PBS+0.1% Tween and washed twice with 50 .mu.l PBS.

[0109] 12.5 .mu.l/well secondary antibody anti-mouse HRP was added to each well at a dilution of 1/5000 in PBS+0.1% Tween+2% milk and incubated for 40 minutes at room temperature. The wells were washed 3.times. with 50 .mu.l of PBS+0.1% Tween and 3 washes with 50 .mu.l PBS.

[0110] Detection: TMB (transparent plates) or ECL (White plates), 25 .mu.l/well TMB (3,3',5,5'-Tetramethylbenzidine) was added. The reaction was stopped by adding 25 .mu.l of sulfuric acid 2N (1 M). A spectrometer reading at DO=450 nm was then taken.

Example 1

Identification of Single Domain Antibodies Recognizing LF Protein

[0111] A series of assays were performed to identify anti-LF single-domain antibodies from a V.sub.HH library, and to validate the anti-LF antibodies by Phage ELISA and V.sub.HH ELISA assays.

[0112] Three rounds of phage display selection were conducted using biotinylated LF protein. A synthetic hydroxysteroid dehydrogenase type 2 (HSD2) single heavy chain variable domain antibody ("V.sub.HH") library of 3.times.10.sup.9 clones (provided by Hybrigenics Services, Paris, France) was expressed at the surface of M13 phage. The phage display allowed the selection of V.sub.HHs recognizing the non-adsorbed antigen in a native form. Selected V.sub.HHs were validated in non-adsorbed phase ELISA and V.sub.HH ELISA assays, as set forth in the schematic of FIG. 4.

[0113] Antigen Preparation:

[0114] A non-related protein (His-SNAP-Halo-Biotin) was used in an initial round of phage display to delete the library from unspecific binding molecules.

[0115] Prior to the phage display selection, LF-Biotin and His-SNAP-Halo-Biotin were bound to streptavidin magnetic beads (DYNABEADS.RTM. M-280 Streptavidin, provided by Life Technologies, Carlsbad, Calif.) with a 50 nM final concentration of biotinylated protein for the first round, and a 10 nM final concentration of biotinylated protein for the second and third rounds. Successful binding of the biotinylated proteins on the streptavidin beads was confirmed in an SDA-PAGE/Western blot using a streptavidin-HRP conjugate (provided by Thermo Fisher Scientific, Waltham, Mass.). FIG. 5 illustrates a Western blot of biotinylated proteins fixed on streptavidin magnetic beads. As shown, Lane 1=1182 ng of LF-Biotin without beads; Lane 2=1182 ng of LF-Biotin on beads; Lane 3=258 ng of LF-Biotin on beads; Lane 4=155 ng of His-SNAP-Halo-Biotin without beads; Lane 5=155 ng of His-SNAP-Halo-Biotin on beads. The Western blot indicated that the biotinylated proteins were successfully bound to the magnetic beads.

[0116] Phage Display Selection (3 Rounds):

[0117] The HS2D Ab Phage Display library presenting 3.times.10.sup.9 V.sub.HHs was first incubated with the His-SNAP-Halo-Biotin beads to remove unspecific binders. The unbound V.sub.HHs expressed as an E. coli supernatant were then incubated with the LF-Biotin beads. A total of three rounds of phage display were performed, as shown in the schematic of FIG. 6. The depletion step was repeated before each Phage Display round to remove non-specific V.sub.HHs.

[0118] To determine the binding of specific anti-LF V.sub.HHs during each round of Phage Display, an output/input ratio was determined by measuring the concentration of specific anti-LF V.sub.HHs before and after 3 consecutive rounds of phage display selection. The results are shown below in Table 2 and are illustrated graphically in FIG. 7. It was determined that the third round exhibited the greatest binding compared to the first and second rounds.

TABLE-US-00002 TABLE 2 Input/Output Values and Ratios During 3 Consecutive Rounds of Phage Display Round 1 Round 2 Round 3 Input 8.1E+11 1.3E+12 1.5E+12 Output 4.9E+5 3.7E+7 2.1E+8 Output/Input 6.1E-07 2.7E-5 1.4E-4

[0119] An ELISA assay on the pool of each round was performed to further determine binding during the 3 consecutive rounds of phase display selection. The results are illustrated graphically in FIG. 8. As shown, progressive binding during the cycle indicates that the Phage Display conditions are appropriate. At the end of the 3rd round of Phage Display, 183 E. coli clones were randomly selected for further analysis.

[0120] V.sub.HH Validation Using Non-Adsorbed Phage ELISA

[0121] The LF-Biotin binding of the 183 V.sub.HH clones selected after three rounds of Phage Display was tested using a non-adsorbed phage ELISA assay that allowed for the proper folding of the native LF protein, as illustrated in the schematic of FIG. 9.

[0122] The phages produced from each E. coli clone were used in a 384 well plate ELISA assay with HRP-conjugated anti-M13 antibody (provided by GE Healthcare, Chicago, Ill.) and a colorimetric substrate (TMB, Tetramethylbenzidine, Thermo Fisher Scientific, Waltham, Mass.). The results of the first ELISA assay with random clones are set forth in Table 3, below. V.sub.HH clones that exhibited a significant ELISA signal in the presence of LF-Biotin and a very low signal in the presence of His-SNAP-Halo-Biotin were considered as specific LF binders and were selected for sequence analysis.

TABLE-US-00003 TABLE 3 Results of Non-Adsorbed ELISA of 183 Randomly Selected Clones No. Total Percentage Positive Hit 30 30 16.4% Weak Positive Hit 0 Negative Hit 153 153 83.6%

[0123] The sequencing of the positive clones revealed the presence of 25 available different positive V.sub.HHs. They were reconfirmed (N=2) by non-adsorbed ELISA, as shown in FIGS. 10a-10f.

[0124] V.sub.HH Validation Using Non-Adsorbed V.sub.HH ELISA

[0125] The LF-Biotin binding of the 25 selected V.sub.HH clones was tested using non-adsorbed V.sub.HH ELISA that allows for the proper folding of the native LF protein and to select the best candidates (FIG. 9).

[0126] The V.sub.HH-myc produced by IPTG induction from each E. coli clone were used in a 384 well plate ELISA with anti-myc (provided by Invitrogen Corp., Carlsbad, Calif.), HRP-conjugated anti-mouse (provided by Novus Biologicals, Littleton, Colo.) and a calorimetric substrate (TMB, Tetramethylbenzidine, Thermo Fisher Scientific, Waltham, Mass.). V.sub.HH clones with a significant ELISA signal in the presence of LF-Biotin and a very low signal in the presence of His-SNAP-Halo-Biotin were considered as specific LF binders and were selected (FIGS. 11a-11f).

[0127] Sequences and Clones Delivery

[0128] 25 clones were selected for further testing. As shown in Table 4, the phage ELISA ratio of signal LF/Signal HIS-SNAP-Halo for all 25 clones were positive. The Induced ELISA ratio of signal LF/signal His-SNAP-Halo for clones 1-13 was positive, clones 14-19 was weakly positive, and for clones 20-25 was negative.

[0129] Clone 16 had an Amber codon and can be corrected because only the Phage can read this codon like a Glutamic Acid. This codon is read like a STOP codon in the other systems (mammals or classical bacterial strain).

TABLE-US-00004 TABLE 4 Summary of Clone Redundancy Phage ELISA Induced ELISA ratio signal ratio Signal Clone LF/signal LF/signal Amber name Redundancy His-SNAP-Halo His-SNAP-Halo Codon 1 3 45.5 6.3 No 2 3 36.5 5.0 No 3 2 47.2 6.3 No 4 1 34.6 9.2 No 5 1 41.5 8.0 No 6 1 8.9 7.3 No 7 1 17.0 6.8 No 8 1 30.2 5.5 No 9 1 38.1 4.6 No 10 1 7.7 3.9 No 11 1 22.6 3.8 No 12 1 21.9 3.7 No 13 1 34.6 3.0 No 14 1 39.0 2.9 No 15 1 46.6 2.9 No 16 1 14.6 2.9 Yes 17 1 28.4 2.7 No 18 1 40.9 2.6 No 19 1 31.6 2.2 No 20 1 28.4 2.0 No 21 1 35.9 1.7 No 22 1 34.9 1.7 No 23 1 46.2 1.6 No 24 1 22.1 1.5 No 25 1 44.5 1.5 No

[0130] 10 clones were collected from the screening according to redundancy, ELISA assay results, and sequencing results. The 10 clones selected were clones 1, 6, 3, 4, 5, 8, 7, 9, 2, and 10. The amino acid sequence of each clone was identified as clone 1 (SEQ ID NO: 1); clone 6 (SEQ ID NO: 2); clone 3 (SEQ ID NO: 3); clone 4 (SEQ ID NO: 4); clone 5 (SEQ ID NO: 5); clone 8 (SEQ ID NO: 6); clone 7 (SEQ ID NO: 7); clone 9 (SEQ ID NO: 8); clone 2 (SEQ ID NO: 9); clone 10 (SEQ ID NO: 10).

[0131] The alignment of the 10 selected clones are shown in FIG. 12. The reference is a nanobody with CFR region replaced by "X."

[0132] A map of V.sub.HHr of pHEN2 is shown in FIG. 13.

Example 2

Identification of Single-Domain Antibodies Recognizing EF Protein

[0133] A series of assays was conducted to identify anti-EF single-domain antibodies from a V.sub.HH library. The anti-EF single domain antibodies were validated by Phage ELISA assay.

[0134] Three rounds of Phage Display selection were conducted using biotinylated EF protein. A synthetic HS2D Ab V.sub.HH library of 3.times.10.sup.9 clones was expressed at the surface of M13 phage. The phage display allowed the selection of V.sub.HHs recognizing the non-adsorbed antigen in a native form. Selected V.sub.HHs were validated in non-adsorbed Phage ELISA.

[0135] Antigen Preparation

[0136] As described in Example 1, His-SNAP-Halo-Biotin was used in an initial round of Phage Display to deplete the library from unspecific binding molecules.

[0137] Prior to the Phage Display selection, EF-Biotin and His-SNAP-Halo-Biotin were bound to streptavidin magnetic beads (Dynabeads.RTM. M-280 Streptavidin, provided by Life Technologies, Carlsbad, Calif.) with a 50 nM final concentration of biotinylated protein for the first round and 10 nM final concentration of biotinylated protein for the second and third rounds. Successful binding of the biotinylated proteins on the streptavidin beads was confirmed in an SOS-PAGE/Western Blot using a Streptavidin-HRP conjugate (provided by Thermo Fisher Scientific, Waltham, Mass.).

[0138] FIG. 14 illustrates a Western blot of biotinylated proteins fixed on Strepavidin magnetic beads. Lane 1: 250 ng of EF-Biotin without beads; Lane 2: 1140 ng of EF-Biotin on beads; Lane 3: 250 ng of EF-Biotin on beads; Lane 4: 155 ng of His-SNAP-Halo-Biotin without beads; Lane 5: 155 ng of His-SNAP-Halo-Biotin on beads.

[0139] Phage Display Selection (3 Rounds)

[0140] The HS2D Ab Phage Display library presenting 3.times.10.sup.9 V.sub.HHs was first incubated with the His-SNAP-Halo-Biotin beads to remove unspecific binders. The unbound V.sub.HHs expressed as an E. coli supernatant were incubated with the EF-Biotin beads. A total of three rounds of Phage Display were performed, as shown in the schematic of FIG. 6. The depletion step was repeated before each Phage Display round to remove non-specific V.sub.HHs.

[0141] To determine binding of the specific anti-EF V.sub.HHs during each round of Phage Display, an output/input ratio was determined by measuring the concentration of specific anti-EF V.sub.HHs before and after the 3 consecutive rounds of phage display selection. The results are shown below in Table 5 and are illustrated graphically in FIG. 15.

TABLE-US-00005 TABLE 5 Input/Output Values and Ratios During 3 Consecutive Rounds of Phage Display Round 1 Round 2 Round 3 Input 8.1E+11 3.5E+11 3.3E+11 Output 3.9E+5 2.6E+8 2.1E+9 Output/Input 4.8E-07 7.5E-4 6.3E-3

[0142] An ELISA assay was performed on the pool of each round to further determine binding during the 3 consecutive rounds of phase display selection. The results are indicated graphically in FIG. 16. As shown, progressive binding during the cycle shows that the Phage Display conditions are appropriate. At the end of the 3rd round of Phage Display, 90 E. coli clones were selected randomly.

V.sub.HH Validation Using Non-Adsorbed Phage ELISA

[0143] The EF-Biotin binding of the 90 V.sub.HH clones selected after three rounds of Phage Display was tested using a non-adsorbed phage ELISA assay that allowed for the proper folding of the native EF protein, as illustrated in the schematic of FIG. 9.

[0144] The phages produced from each E. coli clone were used in a 384 well plate ELISA with HRP-conjugated anti-M13 antibody (provided by GE Healthcare, Chicago, Ill.) and a colorimetric substrate (TMB, Tetramethylbenzidine, Thermo Fisher Scientific, Waltham, Mass.). The results of the first ELISA assay with random clones are set forth below in Table 6. V.sub.HH clones with a significant ELISA signal in the presence of EF-Biotin and a very low signal in the presence of His-SNAP-Halo-Biotin were considered as specific EF binders and were selected for sequence analysis.

TABLE-US-00006 TABLE 6 Results of Non-Adsorbed ELISA of 90 Randomly Selected Clones ID. No. Total Percentage Positive Hit 78 82 91.1% Weak Positive Hit 4 Negative Hit 8 8 8.9%

[0145] The sequencing of the positive clones revealed the presence of 41 available different positive V.sub.HHs. The selected V.sub.HHs were reconfirmed (N=2) by non-adsorbed ELISA, as shown in FIGS. 17a-17g.

[0146] V.sub.HH Validation Using Non-Adsorbed V.sub.HH ELISA

[0147] The EF-Biotin binding of the 41 selected V.sub.HH clones was tested using a non-adsorbed V.sub.HH ELISA that allowed for the proper folding of the native EF protein and to select the best candidates (FIG. 9).

[0148] The V.sub.HH-myc produced by IPTG induction from each E. coli clone were used in a 384 well plate ELISA with anti-myc (provided by Invitrogen Corp.. Carlsbad, Calif.), HRP-conjugated anti-mouse (provided by Novus Biologicals, Littleton, Colo.) and a colorimetric substrate (TMB, Tetramethylbenzidine, Thermo Fisher Scientific, Waltham, Mass.). V.sub.HH clones with a significant ELISA signal in the presence of EF-Biotin and a very low signal in the presence of His-SNAP-Halo-Biotin were considered to be specific EF binders and were selected (FIGS. 18a-g).

[0149] Sequences and Clones Delivery

[0150] 10 clones were selected for further testing based on redundancy, ELISA assay results, and the sequencing results (clones 26, 28, 40, 41, 42, 44, 49, 57, 60, and 66). The phage ELISA Ag+/Ag- and phage IPTG Ag+/Ag- were all positive.

TABLE-US-00007 TABLE 7 Summary of Clone Redundancy Phage ELISA Phage IPTG Clone Redundancy Ag+/Ag- Ag+/Ag- 26 2 51.3 10.2 28 3 44.5 6.6 40 2 31.5 6.2 41 2 36.7 5.7 42 15 36.2 5.0 44 2 52.7 6.1 49 2 36.7 3.6 57 2 40.1 6.3 60 2 43.6 4.9 66 2 51.8 5.8

The alignment of the 10 selected clones are shown in FIG. 19. The amino acid sequence of each clone was identified as clone 26 (SEQ ID NO: 11); clone 28 (SEQ ID NO: 12); clone 40 (SEQ ID NO: 13); clone 41 (SEQ ID NO: 14); clone 42 (SEQ ID NO: 15); clone 44 (SEQ ID NO: 16); clone 49 (SEQ ID NO: 17); clone 57 (SEQ ID NO: 18); clone 60 (SEQ ID NO: 19); clone 66 (SEQ ID NO: 20).

[0151] The reference is a nanobody with CFR region replaced by "X."

Example 3

Identification of Single Domain Antibodies Recognizing TSST-1 Protein

[0152] A series of assays were performed to identify anti-TSST-1 single-domain antibodies from a V.sub.HH library, and to validate the anti-TSST-1 antibodies by non-adsorbed phase ELISA.

[0153] Three rounds of Phage Display selection were carried out using biotinylated TSST1. A synthetic HS2D Ab V.sub.HH library of 3.times.10.sup.9 clones was expressed at the surface of M13 phage. The Phage Display allowed the selection of V.sub.HHs recognizing the non-adsorbed antigen in a native form. Selected V.sub.HHs were validated in non-adsorbed Phage ELISA.

[0154] VHH Selection in vitro and Phase ELISA Validation

[0155] After three rounds of Phage Display, 90 V.sub.HHs were selected and analyzed for binding to TSST1. However, none were positive in non-adsorbed phage ELISA.

[0156] VHH Selection in Yeast

[0157] The coding sequence for S. aureus TSST-1 (aa 41-234) was FOR amplified and cloned into pB27 as a C terminal fusion to LexA (LexA-TSST1), as shown in the schematic of FIG. 20.

[0158] During the First Round of screening, 1.1.times.10.sup.6 nanobodies were collected. A yeast library from Round 1 was created with 2.5.times.10.sup.5 yeast cells complexity. During the 2YH screen against TSST1 bait, 1 million interactions were tested, and 190 positive clones were selected. The sequencing of the clones revealed the presence of about 28 different clones.

[0159] Antigen Preparation

[0160] As described in Example 1, His-SNAP-Halo-Biotin was used in an initial round of Phage Display to deplete the library from unspecific binding molecules.

[0161] Prior to the Phage Display selection, TSST1-Biotin and His-SNAP-Halo-Biotin were bound to Streptavidin Magnetic Beads (Dynabeads.RTM. M-280 Streptavidin, provided by Life Technologies, Carlsbad, Calif.) with a 50 nM final concentration of biotinylated protein for the first round and 10 nM final concentration of biotinylated protein for the second and third rounds. Successful binding of the biotinylated proteins on the streptavidin beads was confirmed in an SOS-PAGE/Western Blot using a Streptavidin-HRP conjugate (provided by Thermo Fisher Scientific, Waltham, Mass.).

[0162] FIG. 21 illustrates a Western blot of biotinylated proteins fixed on Streptavidin magnetic beads. Lane 1: 302 ng of TSST1-Biotin without beads (50 nM); Lane 2: 302 ng of TSST1-Biotin on the beads (50 nM for the Round 1); Lane 3: 66 ng of TSST1-Biotin on beads (10 nM for the rounds 2 and 3); Lane 4: 155 ng of His-SNAP-Halo-Biotin without beads (10 nM for all the rounds); Lane 5: 155 ng of His-SNAP-Halo-Biotin on beads (10 nM for the all the rounds).

[0163] Phage Display Selection

[0164] The HS2D Ab Phage Display library presenting 3.times.10.sup.9 V.sub.HHs was first incubated with the His-SNAP-Halo-Biotin beads to remove unspecific binders. The unbound V.sub.HHs expressed as an E. coli supernatant were incubated with the TSST-1-Biotin beads. A total of three rounds of Phage Display were performed, as shown in the schematic of FIG. 6. The depletion step was repeated before each Phage Display round to remove unspecific V.sub.HHs.

[0165] To determine the binding of specific anti-TSST-1 V.sub.HHs during each round of Phage Display, an output/input was determined by measuring the concentration of specific anti-TSST-1 V.sub.HHs before and after the 3 consecutive rounds of phage display selection. The results are shown below in Table 8 and are illustrated graphically in FIG. 22.

TABLE-US-00008 TABLE 8 Input/Output Values and Ratios During 3 Consecutive Rounds of Phage Display Round 1 Round 2 Round 3 Input 8.1E+11 5.9E+12 1.6E+11 Output 2.1E+5 4.3E+7 2.1E+9 Output/Input 2.6E-07 7.3E-5 1.3E-2

[0166] An ELISA assay on the pool of each round was performed, as shown in FIG. 23. Progressive enrichment during the cycle shows that the Phage Display conditions are appropriate (Table 8). At the end of the 3rd round of Phage Display, 90 E. coli clones were randomly selected and analyzed by non-adsorbed phage ELISA.

[0167] V.sub.HH Validation using Non-Adsorbed Phage ELISA

[0168] The binding of the 90 V.sub.HH selected clones to TSST-1 was tested using anon-adsorbed phage ELISA assay that allows for the proper folding of the native TSST-1 protein, as described in Example 1.

[0169] The phages produced from each E. coli clone were used in a 384 well plate ELISA with HRP-conjugated anti-M13 antibody (provided by GE Healthcare, Chicago, Ill.) and a colorimetric substrate (TMB, TetraMethylBenzidine, Thermo Fisher Scientific, Waltham, Mass.). The first ELISA with random clones are presented in Table 9. V.sub.HH clones with a significant ELISA signal in the presence of TSST-1-Biotin and a very low signal in the presence of His-SNAP-Halo-Biotin were considered as specific TSST-1 binders. As indicated in Table 9, none of the clones were positive for TSST-1 protein. The data is shown graphically in FIG. 24.

TABLE-US-00009 TABLE 9 Results of Non-Adsorbed ELISA of 90 Randomly Selected Clones ID. No. Total Percentage Positive Hit 0 0 0% Weak Positive Hit 0 Negative Hit 90 90 100%

[0170] V.sub.HH Selection

[0171] The coding sequence for S. aureus TSST11 (aa 41-234) was PCR amplified and cloned into pB27 as a C-terminal fusion to LexA (LexA-TSST-1).

[0172] The construct was checked by sequencing the entire insert and used as a bait to screen the Yeast Library from round 1 of phage display. pB27 derives from the original pBTM116 (Vojtek and Hollenberg, 1995) plasmid.

[0173] During the first round of screening, 1.1.times.10.sup.6 nanobodies were collected. A Yeast Library from this round was created with 2.5.times.10.sup.6 yeast cells complexity.

[0174] A test screen with TSST1 using the LexA system was completed. The protein was determined not to be toxic, but it was little auto activating. As a result, a selection medium with 20 mM of 3-Aminotriazol for the final screen. 1 million interactions were tested. In total, 190 positive clones were selected and sequenced, revealing the presence of about 28 different clones (clones 67-94). A summary of the clone redundancy is given in Table 10.

TABLE-US-00010 TABLE 10 Summary of Clone 67- Redundancy Glycosylation Clone ID Redundancy Site 67 28 No 68 20 No 69 12 No 70 11 No 71 10 No 72 8 No 73 7 No 74 6 No 75 5 No 76 5 No 77 4 No 78 3 No 79 3 No 80 2 No 81 1 No 82 1 No 83 1 No 84 1 No 85 1 No 86 1 No 87 1 No 88 1 No 89 1 No 90 1 No 91 22 Yes 92 7 Yes 93 2 Yes 94 1 Yes

[0175] Sequences and Clone Delivery

[0176] 10 of the 28 clones were selected based on redundancy, glycosylation site, and sequencing results (Clones 67, 76, 69, 70, 75, 68, 73, 74, 71, and 72). The amino acid sequence of each clone was identified as clone 67 (SEQ ID NO: 21); clone 76 (SEQ ID NO: 22); clone 69 (SEQ ID NO: 23); clone 70 (SEQ ID NO: 24); clone 75 (SEQ ID NO: 25); clone 68 (SEQ ID NO: 26); clone 73 (SEQ ID NO: 27); clone 74 (SEQ ID NO: 28); clone 71 (SEQ ID NO: 29); clone 72 (SEQ ID NO: 30).

[0177] The 10 selected clones were aligned with a reference nanobody with CDR region replaced by X, as shown in FIG. 25.

Sequence CWU 1

1

601121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 1Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Arg 20 25 30Trp Asp Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Leu Gly Asn Met Asp Ser Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gln Glu Glu Lys Trp Met Ser Pro Gln Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1202126PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 2Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Tyr Ser 20 25 30Ser Asp Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Arg Gln Asn Trp Asp Arg Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Tyr Trp Gly His Gly Thr Tyr Glu Phe Asp Pro Lys 100 105 110Ser Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 1253121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 3Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Tyr 20 25 30Ser Tyr Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Arg Asp Gly Asp Trp Lys Asn Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Phe Glu Glu His Trp Met Thr Pro Leu Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1204121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 4Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg 20 25 30Ile Asp Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Phe Asp Thr Ser His Glu Asn Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Pro Glu Ser Met Trp Phe Ser Ile Glu Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1205121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 5Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Ser Phe 20 25 30Arg Asp Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Trp Asn Glu Gly His Ala His Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Ser Glu Asp Met Trp Phe Asn His Phe Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1206124PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 6Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ser 20 25 30His Tyr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Tyr Asn His Asp His Asp Thr Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gly Ala Thr Ala Thr Thr Asp Gly Gly Tyr Gln Glu 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 1207121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 7Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ser Tyr Ala 20 25 30Asn Ser Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Gly Asp Ser Ala Tyr Tyr Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Thr Gln Gln Ile Gly Met Arg Glu Tyr Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1208121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 8Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Tyr Ser Ala 20 25 30Glu Thr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Asp Pro Thr Thr Thr Thr Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Tyr Gln Arg Leu Asp Asn Phe Glu Ile Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 1209124PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 9Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Phe Ser Asn 20 25 30Ile Glu Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Tyr Glu Asn Glu Ser Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Ala Gln Ser Tyr His Gly Lys Lys Gly Met Thr Glu 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12010124PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 10Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Trp 20 25 30Ser Glu Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Arg Tyr Thr Asp Asp Tyr Ser Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Thr Tyr His Tyr Gln Tyr Gly Gly Asp Gln Thr Phe 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12011121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 11Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Tyr Ser Ala 20 25 30Arg Thr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ala His Glu Gly Gln Ala Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Thr Arg Ala His Glu Pro Lys Gly Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12012121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 12Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Ser Tyr Arg 20 25 30Gly Thr Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Phe Arg Pro Asp Gln Gln Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Glu Arg Arg Gln His Ser Arg Gly Leu Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12013121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 13Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asp 20 25 30Gly Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ala Ser Pro Ser Gly Glu Ser Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gly Arg Gly Arg Ser Gln Lys Ser Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12014121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 14Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Trp Arg 20 25 30Thr Thr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly Gly Asp Asn Arg Gly Thr Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gly Arg Leu Asn Gln Asn Gly Pro Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12015121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 15Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Gly Ser Phe 20 25 30Ala Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ala His Ala Gly Gly Ser Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gln Arg Ile Gly Ala Thr Arg Ala Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12016121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 16Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Gly 20 25 30Ser Thr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Arg Phe Glu Asn Gln Gly Asn Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Thr Arg Ala Val Ala Ser Ser Thr Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12017121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 17Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Phe Phe Thr 20 25 30Ser Ser Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Arg Gln His Asp Asn Ala Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Ala Arg Asn Ala Thr His Lys Gly Ile Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12018121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 18Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Ser Phe 20 25 30Gln Thr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ser Arg Gln Gly Ser His Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Thr Arg Met Thr Glu Thr Arg

Lys Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12019121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 19Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Lys 20 25 30His Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ser Gly Pro Gly Met Glu Ser Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gly Arg Ala Phe Pro Gly Gln Lys Met Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12020130PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 20Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Gly Ser Asn 20 25 30Ala Ser Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Arg His Glu Gly Gln Pro Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Met Arg Tyr Gln Ser His Ile Asp Ile Pro Gly Asp 100 105 110Ser Ser Pro Lys Leu Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125Ser Ser 13021127PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 21Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Ser Gly 20 25 30Ala Thr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ser Trp Ala Ser Ile Trp Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Tyr Lys Leu Tyr Gly Asp Met Thr Met Glu Trp Gly 100 105 110Phe Lys Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 12522121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 22Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Ser Asn 20 25 30Arg Asp Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Gly His Gly Ser Thr Gln Gly Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Glu Leu Asp Arg Glu Trp Gly Lys Trp Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12023121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 23Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Gly 20 25 30Gln Asp Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ser Tyr Ser Ser Val Asp Gly Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Ile Ile Asp Glu Gln Phe His Met Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12024121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 24Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Tyr Ser Glu 20 25 30Glu Thr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Phe Ala Gln Asp Gly Tyr Arg Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Glu Phe Asp Asn Val Trp His Leu Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12025121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 25Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Ala 20 25 30Glu Glu Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ala Gln Gln Gly Ala Glu Pro Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Glu Phe Asp Asn Val Trp His Leu Pro Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12026124PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 26Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Ser Trp 20 25 30Arg Glu Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Tyr Gly Trp Asp Ser Val Gly Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Ile Glu Thr Tyr Gly Ala Ser Asn Gly Trp Arg Glu 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12027127PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 27Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Tyr Phe Thr 20 25 30Ile Glu Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Phe Trp Trp Thr Asn Thr His Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Pro Met Thr Glu Arg Ala Glu Ala Gln Met Gly Thr 100 105 110Met Glu Pro Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 12528121PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 28Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Tyr Ser Lys 20 25 30Glu Thr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Asp Trp Pro Asp Tyr Tyr Gly Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Trp Val Asn Glu Lys Phe Gly Tyr Val Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12029124PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 29Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala Thr Ser Lys 20 25 30Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Ser Trp Asn Asn Asp Trp Ser Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Tyr Asp Leu Gln His Gly Ser Gly Met Pro Arg Met 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12030130PRTArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 30Met Ala Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Phe Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Glu 20 25 30Ile Thr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45Phe Val Ser Ala Ile Ser Glu Trp Pro Gly Asn Tyr Ala Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn65 70 75 80Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr 85 90 95Tyr Tyr Cys Ala Gly Asp Gly Leu Ala Ser Asp Phe Tyr Gly Lys Ile 100 105 110Asp Glu Leu Asp Met Met Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 125Ser Ser 13031363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 31atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagatact tatcgttggg ataacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtctggg taacatggac 180tcgtactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300caggaggaga agtggatgtc gcctcagtac tggggacagg ggacgcaggt aactgtgagt 360agc 36332378DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 32atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaacaaca tatagttcgg acacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtcgtca aaactgggac 180cgttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300tattggggtc acggtacata tgagtttgac cctaagtcga tgtattgggg acaggggacg 360caggtaactg tgagtagc 37833363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 33atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcaact tcatattcgt acaacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgcgtgacgg tgactggaag 180aactactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300tttgaggagc actggatgac acctctgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36334363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 34atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggacgtaca tttcgtatcg acacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgtttgacac aagtcatgag 180aactattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300cctgagtcga tgtggttttc gattgagtat tggggacagg ggacgcaggt aactgtgagt 360agc 36335363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 35atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcacaact agttttcgtg acacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgtggaacga agggcatgca 180cattactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300tcggaggaca tgtggtttaa ccacttttat tggggacagg ggacgcaggt aactgtgagt 360agc 36336372DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 36atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcaact agtagtcatt acgacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgtacaacca tgaccatgac 180acatactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300ggtgcaacag caacaacaga cggtggttat caggagtatt ggggacaggg gacgcaggta 360actgtgagta gc 37237363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 37atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaacatcc tacgcaaaca gtgacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtggtga cagtgcatat 180tactattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300acacagcaga ttggtatgcg tgagtattat tggggacagg ggacgcaggt aactgtgagt 360agc 36338363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 38atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcatac agtgcagaga cagacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtgaccc tacaacaaca 180acatactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300tatcagcgtc tggacaactt tgagatttat tggggacagg ggacgcaggt aactgtgagt 360agc 36339372DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 39atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcttcttc tcaaacatcg aggacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggttatga aaacgagtcg 180ccttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc

tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300gcacagtcgt atcacggtaa gaagggtatg acagagtatt ggggacaggg gacgcaggta 360actgtgagta gc 37240372DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 40atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggacgtaca tcatggtcgg aaattatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgcgttatac agacgactat 180tcgtactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300acatatcact atcagtatgg tggtgaccag acattttact ggggacaggg gacgcaggta 360actgtgagta gc 37241363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 41atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagcatac agtgcacgta caacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct ccgcacatga agggcaggca 180ccttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300acacgtgcac acgagcctaa gggtatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36342363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 42atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagcatcc tatcgtggta catctatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cctttcgtcc tgaccagcag 180ccttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300gagcgtcgtc agcactcgcg tggtctgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36343363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 43atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggacgtaca ttcgacggtt ccgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cggcatcgcc ttcgggtgag 180tcgtattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300ggtcgtggtc gttcgcagaa gtcgatgtat tggggacagg ggacgcaggt aactgtgagt 360agc 36344363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 44atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagataca tggcgtacaa ctgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtggtga caaccgtggt 180acatattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300ggtcgtctga accagaacgg tcctatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36345363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 45atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagatgga tcatttgcat ccgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cggcacatgc aggtggttcg 180ccttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300cagcgtattg gtgcaacacg tgcaatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36346363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 46atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcttcaca agtggttcga cagcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgcgttttga aaaccagggt 180aactattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300acacgtgcag ttgcatcgtc gacaatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36347363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 47atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaggcttt tttacatcga gtacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cccgtcaaca tgacaacgca 180ccttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240atggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300gcacgtaacg caacacacaa gggtatttac tggggacagg ggacgcaggt aactgtgagt 360agc 36348363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 48atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcggcaca tcatttcaga cagcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgagtcgtca aggttcgcat 180ccttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300acacgtatga cagagacacg taagatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36349363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 49atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcaact tttaagcatt ccgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgagtggtcc tggtatggag 180tcgtactatg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300ggtcgtgcat ttcctggtca gaagatgtac tggggacagg ggacgcaggt aactgtgagt 360agc 36350390DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 50atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaacaggc tcaaacgcat ccgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cccgtcatga aggtcagcct 180ccttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300atgcgttatc agtcgcacat tgacattcct ggtgactcgt cgcctaagct gatgtattgg 360ggacagggga cgcaggtaac tgtgagtagc 39051381DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 51atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcgatact agtggtgcaa cagtaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct ccagttgggc aagtatttgg 180ccttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300tataagctgt atggtgacat gacaatggag tggggtttta agtcgtattg gggacagggg 360acgcaggtaa ctgtgagtag c 38152363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 52atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagatact agtaaccgtg acgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgggtcatgg tagtacacag 180ggttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300gagctggacc gtgagtgggg taagtggtac tggggacagg ggacgcaggt aactgtgagt 360agc 36353363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 53atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcaaca tacggtcagg atacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgagttattc gtcggttgac 180ggttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300attattgacg agcagtttca catgccttat tggggacagg ggacgcaggt aactgtgagt 360agc 36354363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 54atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggcacatac tcagaggaga ctgcaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgtttgcaca agacggttat 180cgttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300gagtttgaca acgtttggca cctgccttat tggggacagg ggacgcaggt aactgtgagt 360agc 36355363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 55atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggatcaaca agtgcagagg agtctatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cggcacaaca aggtgcagag 180ccttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300gagtttgaca acgtttggca cctgccttat tggggacagg ggacgcaggt aactgtgagt 360agc 36356372DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 56atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaacaaca tcatggcgtg agacaatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgtacggttg ggactcggtt 180ggttattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300attgagacat atggtgcatc gaacggttgg cgtgagtact ggggacaggg gacgcaggta 360actgtgagta gc 37257381DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 57atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaggctat ttcacaatcg aggacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct ccttttggtg gacgaacaca 180cattactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgcc 300cctatgacag agcgtgcaga ggcacagatg ggtacaatgg agccttactg gggacagggg 360acgcaggtaa ctgtgagtag c 38158363DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 58atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggaacatat agtaaggaga ctgacatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct ccgactggcc tgactattat 180ggttactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300tgggttaacg agaagtttgg ttatgtttat tggggacagg ggacgcaggt aactgtgagt 360agc 36359372DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 59atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggagcaact tcaaagtcgt acggtatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cgagttggaa caacgactgg 180tcgtactacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300tatgacctgc agcacggttc gggtatgcct cgtatgtatt ggggacaggg gacgcaggta 360actgtgagta gc 37260390DNAArtificial SequenceDerived from a cartilaginous fish heavy chain antibody or a camelid heavy chain nanobody 60atggcggaag tgcagctgca ggcttccggg ggaggatttg tgcagccggg ggggtcattg 60cgactgagct gcgccgcatc cggctcaaca tcagagatca caggtatggg ctggtttcgt 120caggcccctg gcaaggagag agagttcgtt tccgccatct cggaatggcc tggtaactat 180gcatattacg ctgacagcgt aaagggaaga tttacaatta gccgggataa ctccaaaaac 240acggtctatc tccagatgaa cagcctcagg gccgaggaca cagctacgta ttactgtgca 300ggtgacggtc tggcatcgga cttttatggt aagattgacg agctggacat gatgtactgg 360ggacagggga cgcaggtaac tgtgagtagc 390

Claims

1. A nanobody comprising a V.sub.HH domain of a camelid heavy-chain antibody or a VNAR domain of a cartilaginous fish heavy chain antibody, wherein the nanobody is capable of neutralizing one or more bacterial toxins.

2. The nanobody of claim 1, wherein the nanobody is an anti-LF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 1-10.

3. The nanobody of claim 1, wherein the nanobody is an anti-EF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 11-20.

4. The nanobody of claim 1, wherein the nanobody is an anti-TSST-1 nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 21-30.

5. The nanobody of claim 1, wherein the bacterial toxins are associated with anthrax exposure or toxic shock syndrome.

6. The nanobody of claim 1, wherein the nanobody is humanized.

7. A pharmaceutical composition comprising or consisting essentially of a nanobody comprising a V.sub.HH domain of a camelid heavy-chain antibody or a VNAR domain of a cartilaginous fish heavy chain antibody, wherein the nanobody is capable of neutralizing one or more bacterial toxins.

8. The pharmaceutical composition of claim 7, wherein the nanobody is an anti-LF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 1-10.

9. The pharmaceutical composition of claim 7, wherein the nanobody is an anti-EF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 11-20.

10. The pharmaceutical composition of claim 7, wherein the nanobody is an anti-TSST-1 nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 21-30

11. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 7, wherein the bacterial toxins are associated with anthrax exposure or toxic shock syndrome.

13. The pharmaceutical composition of claim 7, wherein the nanobody is humanized.

14. A method of treating, preventing, or alleviating a disorder mediated by one or more bacterial toxins, the method comprising administering an effective amount of a nanobody comprising a V.sub.HH domain of a camelid heavy-chain antibody or a VNAR domain of a cartilaginous fish heavy chain antibody in an amount sufficient to neutralize the bacterial toxins.

15. The method of claim 14, wherein the nanobody is an anti-LF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 1-10.

16. The method of claim 14, wherein the nanobody is an anti-EF nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 11-20.

17. The method of claim 14, wherein the nanobody is an anti-TSST-1 nanobody comprising at least 85% sequence identity to the amino acid sequence set forth in one or more of SEQ ID NO: 21-30.

18. The method of claim 14, wherein the bacterial toxins are associated with anthrax exposure or toxic shock syndrome.

19. A polypeptide comprising or consisting of one or more of SEQ ID NO: 1-30.